1. Field of the Invention
The present invention relates to a method for preparing an R- or S-form xcex1-substituted heterocyclic carboxylic acid (hereinafter referred to as xe2x80x9cxcex1-HCCAxe2x80x9d) and a counter enantiomeric form of xcex1-HCCA ester. More particularly, the present invention pertains to a method for preparing R- or S-form xcex1-HCCA and S- or R-form xcex1-HCCA ester, respectively using an enzyme catalyst with enantioselectivity from a racemate of xcex1-HCCA ester obtained by reacting a racemic xcex1-HCCA and alcohol.
2. Description of the Prior Art
Divided into optical isomers, R- and S-form, tetrahydro-2-furoic acid (hereinafter referred to as xe2x80x9cTHFAxe2x80x9d), a kind of xcex1-HCCA, is an important chiral building block which has various applications in chemistry. Of the optical isomers, R-(+)-THFA is used as a side chain intermediate for the synthesis of penem type antibiotics while S-(xe2x88x92)-THFA is useful as a chiral intermediate for organic synthesis. Thus, THFA is different in use from R form to S form. However, because THFA is obtained in the form of racemate when chemically synthesized, additional processes are required to separate THFA into enantiomers thereof: R and S forms.
Optical resolution has been usually used to divide racemic THFA into R- and S-forms thereof. In 1983, Belanger successfully separated THFA racemate into enantiomers thereof by use of brucine and ephedrine as resolving agents (Can. J. Chem., 61, 1383 (1983)). However, the resolving agents are not economical because of their being very expensive. Another problem with this process is low in enantiomeric excess value.
Japanese Pat. Laid-Open Publication No. 89-216983 discloses the use of a chiral amine(1-(4-halogenophenyl)ethylamine) as a resolving agent, in which diastereomer salts are prepared from R,S-THFA and optically resolved. This method is also economically unfavorable owing to the high price of the chiral amine. Additionally, only low production yields can be obtained because the amount of R,S-THFA to be added in the early reaction is limited to as low as 4 mmol. Furthermore, the chiral THFA finally obtained is poor in enantiomeric excess value.
Japanese Pat. Laid-Open Publication. No. 97-71576 refers to a method of synthesizing R- or S-THFA by treating R- or S-THFA salts with hydrogen halide, which is different from optical resolving methods.
It has been well known for some time that racemates could be optically resolved using enzyme catalysts, such as esterases, lipases, and proteases, to enantioselectively hydrolyze one of the two enantiomers present. For example, U.S. Pat. No. 5,928,933 discloses an enzyme with an enantiomeric excess value of 95% as a result of extensive experiments for reaction specificity of 44 enzymes, including proteases, lipases and esterases. The enzyme catalyst is very useful for the separation of enantiomeric racemates, but because the selectivity for enantiomers and the optical purity of products may vary depending on the choice of enzyme and the chemical structures of substrates, intensive efforts are required to find combinations of enzymes suitable for substrates. Especially, nowhere is found a method for optical resolution of xcex1-HCCA using an enzyme.
Leading to the present invention, the intensive and through research on the optical resolution of xcex1-HCCA, conducted by the present inventors aiming to develop an optically highly pure xcex1-HCCA and a counter enantiomeric form of xcex1-HCCA ester thereto by an economical procedure, resulted in the finding that some of microorganism- or animal-derived hydrolyzing enzymes may enantioselectively hydrolyze the ester functionality of particular optical isomers of xcex1-HCCA esters at high efficiency.
Therefore, it is an object of the present invention to overcome the above problems encountered in prior arts and to provide a method for preparing a highly pure R- or S-form xcex1-HCCA and a counter enantiomeric form of xcex1-HCCA ester thereto using an enzyme, which is economically favorable.
Based on the present invention the above object could be accomplished by providing a method for preparing an R- or S-form xcex1-HCCA and a counter enantiomeric form of xcex1-HCCA ester thereto, comprising the steps of:
reacting a racemic xcex1-HCCA with alcohol to give a racemic xcex1-HCCA ester represented by the following chemical formula 1: 
wherein R1 is selected from the group consisting of substituted or unsubstituted alkyl or alkenyl containing 1 to 6 carbon atoms, benzyl, cycloalkyl containing 3 to 6 carbon atoms, substituted or unsubstituted arylalkyl, and substituted or unsubstituted heteroarylalkyl, X represents O, S or Nxe2x80x94H, and n is an integer of 1 to 3;
optically resolving the racemate of the formula 1 by use of an enzyme with enantioselectivity to hydrolyze either R-form or S-form of the racemate, thereby producing a pure R-form or S-form of xcex1-HCCA and a counter enantiomeric form of xcex1-HCCA ester thereto, said enzyme existing as a powder or an aqueous solution; and
extracting the unhydrolyzed xcex1-HCCA ester with an organic solvent.
The present invention is characterized by the enantioselective hydrolysis of esters of racemic xcex1-HCCA by an enzyme to produce a certain enantiomeric form of xcex1-HCCA and a counter enantiomeric form of the esters of xcex1-HCCA, at once. The separation of the hydrolyzed xcex1-HCCA and the remaining esters of xcex1-HCCA can be achieved by extracting with an organic solvent.
In detail, xcex1-HCCA is reacted with an alcohol at an equivalent amount to produce an xcex1-HCCA ester, which is then enantioselectively hydrolyzed at a constant temperature and pH in an aqueous solution in the presence of an enzyme with enantioselectivity. As a result, the reaction produces an R- or S-form xcex1-HCCA, along with the ester of xcex1-HCCA which has an enantiomeric form counter to that of the hydrolyzed xcex1-HCCA. After the completion of the enantioselective hydrolysis, addition of an organic solvent extracts the ester of xcex1-HCCA thereinto, leaving the xcex1-HCCA in the aqueous phase only. Removal of the organic solvent from the organic phase results in acquisition of an optically pure S- or R-form of xcex1-HCCA ester. Poor in optical purity, the xcex1-HCCA remaining in the aqueous solution may be increased in purity through a purification process using, for example, a column, or may be reused as a starting material in the present invention.
Using a non-enantioselective enzyme or a palladium catalyst, the S- or R-form of xcex1-HCCA ester obtained can be hydrolyzed to an S- or R-form of xcex1-HCCA with a high enantiomeric excess value ( greater than 99%). Additionally, the S- or R-form of xcex1-HCCA ester may be reduced to a chiral alcohol which is useful as an intermediate for the synthesis of various medicines.
For instance, the enantiomeric xcex1-HCCA ester is hydrolyzed at a constant pH and temperature in an aqueous solution in the presence of an enzyme that shows no enantioselectivity and non-specifically hydrolyzes xcex1-HCCA ester. After completion of the enzymatic hydrolysis, the aqueous layer is controlled to pH 2-3 with hydrochloric acid and extracted several times with an organic solvent to yield an S- or R-form of xcex1-HCCA. In the case of an palladium catalyst (Pd/C), the obtained S- or R-form of xcex1-HCCA ester is dissolved in an organic solvent and subjected to hydrogenation at a constant temperature under a predetermined partial hydrogen pressure to produce an S- or R-form of xcex1-HCCA with a high optical purity ( greater than 99%).
In accordance with a preferred embodiment of the present invention, THFA, which belongs to an xcex1-HCCA, is reacted with alcohol at an equivalent amount to give a THFA ester adduct which is then subjected to optical resolution in the presence of an enantioselectively hydrolyzing enzyme to afford an R- or S-form of THFA while leaving a counter enantiomeric form of the THFA ester, which is extracted with an organic solvent. Using a non-enantioselective enzyme or a palladium catalyst, this enantiomeric THFA ester can be returned to an enantiomeric form of THFA with a high optical purity ( greater than 99%). Aside from THFA, all materials falling within the scope of xcex1-HCCA, for example, proline and tetrahydrothiopen-2-carboxylic acid can be optically resolved in accordance with the present invention.
Useful in the present invention are linear or branched alcohols containing 1-6 carbon atoms, aromatic alcohols, cycloalkyl alcohols containing 3-6 carbon atoms, substituted or unsubstituted arylalkyl alcohols, and substituted or unsubstituted heteroarylalkyl alcohols. Preferred are linear alcohols containing 4 or more carbon atoms or aromatic alcohols, when consideration is taken of reaction time and optical purity.
For use in the enantioselective hydrolysis of xcex1-HCCA ester, the enzyme is preferably selected from the group consisting of lipases, proteases, and esterases, all of which are derived from microorganisms or animals. Depending on enzymes, the conformation of the xcex1-HCCA hydrolyzed is determined. Such an enantioselective enzyme, when used, may be in a form of a powder or an aqueous solution. The enzyme is preferably used in an amount of 0.1 to 100 parts by weight based on 100 parts by weight of the xcex1-HCCA ester. For example, if the enzyme amount is less than 0.1 part by weight, the hydrolysis may require excessive time to complete. On the other hand, an enzyme amount exceeding 100 parts by weight increases the production cost.
The enzymatic reaction is optimally carried out at 0-60xc2x0 C. and pH 4-12. As for the organic solvent to extract the remaining enantiomeric xcex1-HCCA ester, it is preferably selected from the group consisting of ethyl acetate, dichloromethane, chloroform, carbon tetrachloride, toluene and mixtures thereof.
Turning now to the reduction of the prepared enantiomeric xcex1-HCCA ester to a corresponding conformation of xcex1-HCCA, a palladium catalyst is preferably used in an amount of 0.1 to 30% by weight and more preferably in an amount of 0.5 to 10% by weight. For example, an amount less than 0.1% by weight is insufficient to perform the hydrogenation. On the other hand, an amount larger than 30% by weight has negative influence on the production cost. At this time, the catalytic hydrogenation of the enantiomeric xcex1-HCCA ester is preferably carried out at a hydrogen partial pressure of 1 to 10 Bars and more preferably at a hydrogen partial pressure of 1 to 5 Bars. For example, the hydrogenation, when being carried out at a hydrogen partial pressure less than 1 bar, is significantly deteriorated in efficiency. On the other hand, a hydrogen partial pressure larger than 10 Bars results in a lot of side products. Other conditions are set at 1 to 20 hours and preferably at 1 to 8 hours for reaction time and at 0 to 70xc2x0 C. and preferably at 20 to 40xc2x0 C. for reaction temperature.
A better understanding of the present invention may be obtained in light of the following examples which are set forth to illustrate, but are not to be construed to limit the present invention.